Team:Peking/Notebook/XTeng
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond"> Xin Teng's Notes</font></font></font> | <font size=6><font color=#585858><font face="Franklin Gothic Demi Cond"> Xin Teng's Notes</font></font></font> | ||
- | <br> <a href="https://2010.igem.org/Team:Peking/Team/XTeng"><img src="https://static.igem.org/mediawiki/2010/9/9c/TX11.jpg" width="40px" alt="goto her page" </a> | + | <br> <a href="https://2010.igem.org/Team:Peking/Team/XTeng"><img src="https://static.igem.org/mediawiki/2010/9/9c/TX11.jpg" width="40px" alt="goto her page" id="imggreen"></a> |
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+ | After carefully studied the structure of MerR, I constructed MerR-MBP (Metal Binding Peptide). Additionaly, I helped to construct and standardize the periplasmic metal binding peptide – DsbA-MerR-MBP using a similar strategy as MerR-MBP. After inductive expression of MerR-MBP-His6, DsbA-PbrR-MBP-His6 and DsbA-MerR-MBP-His6, I use SDS-PAGE and western blot to confirm the expression and localization of MBP and DsbA-MBP. | ||
- | + | There are many pictures and graphs in my notes, I strongly recommand you to download them for more precise reading. | |
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- | < | + | <html> |
- | < | + | <img src="https://static.igem.org/mediawiki/2010/9/91/PKU_Adobe_Reader_Logo.jpg" width=20> |
- | + | <a href="https://static.igem.org/mediawiki/2010/2/22/Note-xteng.pdf"><font color=#FFFFFF>download her notes</font></a></html> | |
- | + | ||
- | <a href="" | + | |
- | </ | + | |
+ | =='''Contents'''== | ||
+ | * <span style="font-size:4mm;">[[Team:Peking/Notebook/XTeng#June| June, 2010]]</span> | ||
+ | |||
+ | * <span style="font-size:4mm;">[[Team:Peking/Notebook/XTeng#July| July, 2010]]</span> | ||
+ | |||
+ | * <span style="font-size:4mm;">[[Team:Peking/Notebook/XTeng#August| August, 2010]]</span> | ||
+ | |||
+ | * <span style="font-size:4mm;">[[Team:Peking/Notebook/XTeng#September| September, 2010]]</span> | ||
+ | |||
+ | * <span style="font-size:4mm;">[[Team:Peking/Notebook/XTeng#October| October, 2010]]</span> | ||
+ | |||
+ | ==June== | ||
+ | ===6.7-6.13=== | ||
+ | |||
+ | Primer design | ||
+ | |||
+ | *For expression test | ||
+ | |||
+ | pET21a-MBP-His | ||
+ | |||
+ | pET 39b DsbA-MBP | ||
+ | |||
+ | pMAL p4X-MBP-MBP | ||
+ | |||
+ | pET21a-MerR-His | ||
+ | |||
+ | pET 39b DsbA-MerR | ||
+ | |||
+ | pMAL p4X-MBP-MerR | ||
+ | |||
+ | *For standard part | ||
+ | |||
+ | MBP_SD | ||
+ | |||
+ | DsbA-MBP_SD | ||
+ | |||
+ | MBP-MBP_SD | ||
+ | |||
+ | MerR_SD | ||
+ | |||
+ | DsbA-MerR_SD | ||
+ | |||
+ | MBP-MerR_SD | ||
+ | |||
+ | ===6.14-6.20=== | ||
+ | Literature Review & Experimental Design for Periplasmic MBP | ||
+ | |||
+ | |||
+ | ==July== | ||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| 1 | ||
+ | |style="text-align:center"| 2 | ||
+ | |style="text-align:center"|3 | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.4-7.11|4]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.4-7.11|5]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.4-7.11|6]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.4-7.11|7]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.4-7.11|8]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.4-7.11|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.4-7.11|10]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.4-7.11|11]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.12-7.18|12]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.12-7.18|13]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.12-7.18|14]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.12-7.18|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.12-7.18|16]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.12-7.18|17]] | ||
+ | |- | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.12-7.18|18]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.19-7.25|19]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.19-7.25|20]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.19-7.25|21]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.19-7.25|22]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.19-7.25|23]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.19-7.25|24]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.19-7.25|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|26]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|27]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|28]] | ||
+ | |style="text-align:center"| 29 | ||
+ | |style="text-align:center"| 30 | ||
+ | |style="text-align:center"| 31 | ||
+ | |- | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | ===7.5-7.11=== | ||
+ | |||
+ | DsbA-MerR (Periplasmic MerR) construction | ||
+ | |||
+ | Successful clones have been verified by sequencing. | ||
+ | |||
+ | |||
+ | Standardization for DsbA-MerR | ||
+ | |||
+ | Double digest shows the right result. Need to be sequenced. | ||
+ | |||
+ | |||
+ | MBP-His (cytosol MBP for expression test) construction | ||
+ | |||
+ | Double digest result shows that clone have failed. No band of right molecular weight can be detected. | ||
+ | |||
+ | MBP (cytosol MBP for mercury binding test) construction | ||
+ | |||
+ | Clones have been constructed, but need to be verified by double digestion. | ||
+ | |||
+ | Mercury Semiquantative Detection | ||
+ | |||
+ | Solution Preparation | ||
+ | |||
+ | 1 g/L Dithizone Cloroform solution | ||
+ | |||
+ | 1.038×10-3 mol/L HgCl2 | ||
+ | |||
+ | Dithizone Eluant | ||
+ | |||
+ | Na2S detoxic solution | ||
+ | |||
+ | 1 mol/L NaOH | ||
+ | |||
+ | Concentrated HNO3 | ||
+ | |||
+ | Standard Mercury Concentration Determination | ||
+ | |||
+ | Mercury concentrated 25 times | ||
+ | |||
+ | Color change can be easily seen, but out of linear range of the standard curve. | ||
+ | |||
+ | ===7.12-7.18=== | ||
+ | DsbA-MBP (periplasmic MBP for expression test) construction | ||
+ | |||
+ | Band of expected molecular weight can be detected by double digest. Need to be sequenced. | ||
+ | |||
+ | Re-design the construction strategy for DsbA-MerR(MBP) | ||
+ | |||
+ | |||
+ | MBP-His & MBP construction | ||
+ | |||
+ | Failed. | ||
+ | |||
+ | Mercury Semiquantative Determination | ||
+ | |||
+ | Standard solution concentrated 10 times. | ||
+ | |||
+ | There is no significant difference between detected mercury concentration prepared in LB or water. But the absolute value is too low. | ||
+ | |||
+ | ===7.19-7.25=== | ||
+ | DsbA-MerR construction. Need to be verified. | ||
+ | |||
+ | DsbA-MBP construction. Need to be verified. | ||
+ | |||
+ | |||
+ | SDS-PAGE & Western Blot | ||
+ | |||
+ | Buffer preparation | ||
+ | |||
+ | 5 × SDS-PAGE buffer | ||
+ | |||
+ | Tris – HCl pH 6.8 | ||
+ | |||
+ | Tris – HCl pH 8.8 | ||
+ | |||
+ | 30% Acr / Bis-Acr | ||
+ | |||
+ | 5 × TBS | ||
+ | |||
+ | 5 × Transfer Buffer | ||
+ | |||
+ | 1 × TTBS | ||
+ | |||
+ | SDS-PAGE result of DsbA-MerR | ||
+ | |||
+ | Overexpression band can be detected at the expected molecular weight (40 kD). | ||
+ | |||
+ | ===7.26-7.28 & 8.3-8.8=== | ||
+ | Double digest verification for DsbA-MBP, MBP-His & MBP. | ||
+ | |||
+ | Failed. | ||
+ | |||
+ | Check the template of MBP by sequencing. | ||
+ | |||
+ | Re-design for MBP & DsbA-MBP construction. | ||
+ | |||
+ | ==August== | ||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| 1 | ||
+ | |style="text-align:center"| 2 | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|3]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|4]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|5]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|6]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|7]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|8]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#8.1-8.15|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.1-8.15|10]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.1-8.15|11]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.1-8.15|12]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.1-8.15|13]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.1-8.15|14]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.1-8.15|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-8.22|16]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-8.22|17]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-8.22|18]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-8.22|19]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-8.22|20]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-8.22|21]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-8.22|22]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.23-8.29|23]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.23-8.29|24]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-9.5|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.23-8.29|26]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.23-8.29|27]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.23-8.29|28]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-9.58.23-8.29|29]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.30-9.5|30]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.16-9.5|31]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | ===8.9-8.15=== | ||
+ | MBP-His construction | ||
+ | |||
+ | Sequencing. | ||
+ | |||
+ | MBP construction | ||
+ | |||
+ | Sequencing. | ||
+ | |||
+ | DsbA-MBP construction | ||
+ | |||
+ | strategy I | ||
+ | |||
+ | Sequencing. | ||
+ | |||
+ | Strategy II | ||
+ | |||
+ | Sequencing. | ||
+ | |||
+ | Standardization for DsbA-MerR | ||
+ | |||
+ | Sequencing. | ||
+ | |||
+ | Standardization for DsbA-MBP | ||
+ | |||
+ | DsbA-MerR sample preparation for SDS-PAGE | ||
+ | |||
+ | ===8.16-8.22=== | ||
+ | DsbA-MerR standardization finished. | ||
+ | |||
+ | Re-construct DsbA-MBP | ||
+ | |||
+ | Expression condition optimization for DsbA-MerR | ||
+ | |||
+ | |||
+ | ===8.23-8.29=== | ||
+ | SDS-PAGE & Western Blot for DsbA-MerR | ||
+ | |||
+ | |||
+ | SDS-PAGE & Western Blot for DsbA-MBP | ||
+ | |||
+ | The ratio of the primary antigen to secondary antigen should be optimized to get better figures. | ||
+ | |||
+ | ===8.30-9.5=== | ||
+ | SDS-PAGE for T3 polymerase | ||
+ | |||
+ | SDS-PAGE & Western Blot for MBP | ||
+ | |||
+ | The ratio of the primary antigen to secondary antigen should be optimized to get better figures. | ||
+ | |||
+ | |||
+ | |||
+ | ==September== | ||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.30-9.5|1]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#8.30-9.5|2]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#8.30-9.5|3]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#8.30-9.5|4]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#8.30-9.5|5]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.6-9.12|6]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.6-9.12|7]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.6-9.12|8]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.6-9.12|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.6-9.12|10]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.6-9.12|11]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.6-9.12|12]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.13-9.19|13]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.13-9.19|14]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.13-9.19|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.13-9.19|16]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.13-9.19|17]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.13-9.19|18]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.13-9.19|19]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.20-9.26|20]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.20-9.26|21]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.20-9.26|22]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.20-9.26|23]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.20-9.26|24]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.20-9.26|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.20-9.26|26]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.27-10.3|27]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.27-10.3|28]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.27-10.3|29]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#9.27-10.3|30]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | |||
+ | ===9.6-9.12=== | ||
+ | Western Blot for MBP | ||
+ | |||
+ | Western Blot for DsbA-MBP | ||
+ | |||
+ | |||
+ | ===9.13-9.19=== | ||
+ | SDS-PAGE & Western Blot for lead DsbA-MBP | ||
+ | |||
+ | |||
+ | SDS-PAGE for lead MBP | ||
+ | |||
+ | The molecular weight of the overexpression bands does not meet our expectation. Check the plasmid. | ||
+ | |||
+ | Scan the spectrum of Crt EBI | ||
+ | |||
+ | ===9.20-9.26=== | ||
+ | Characterize the mutation of PmerT. | ||
+ | |||
+ | |||
+ | ===9.27-10.3=== | ||
+ | 1-18I-pag, 2-2M-pag, 1-18E-pag construction | ||
+ | |||
+ | ==October== | ||
+ | |||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"|- | ||
+ | |style="text-align:center"|- | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.27-10.3|1]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.27-10.3|2]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#9.27-10.3|3]] | ||
+ | |- | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#10.4-10.10|4]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#10.4-10.10|5]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#10.4-10.10|6]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/XTeng#10.4-10.10|7]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#10.4-10.10|8]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/XTeng#10.4-10.10|9]] | ||
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+ | ===10.4-10.10=== | ||
+ | western blot for mercury Lpp-OmpA-MBP | ||
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+ | ===10.11-10.17=== | ||
+ | Cigarette digestion for our biokit. | ||
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+ | Failed. | ||
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<div id="project description bottom"> | <div id="project description bottom"> |
Latest revision as of 09:58, 27 October 2010
There are many pictures and graphs in my notes, I strongly recommand you to download them for more precise reading.
Contents
June
6.7-6.13
Primer design
- For expression test
pET21a-MBP-His
pET 39b DsbA-MBP
pMAL p4X-MBP-MBP
pET21a-MerR-His
pET 39b DsbA-MerR
pMAL p4X-MBP-MerR
- For standard part
MBP_SD
DsbA-MBP_SD
MBP-MBP_SD
MerR_SD
DsbA-MerR_SD
MBP-MerR_SD
6.14-6.20
Literature Review & Experimental Design for Periplasmic MBP
July
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
- | - | - | - | 1 | 2 | 3 |
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
[TOP]
7.5-7.11
DsbA-MerR (Periplasmic MerR) construction
Successful clones have been verified by sequencing.
Standardization for DsbA-MerR
Double digest shows the right result. Need to be sequenced.
MBP-His (cytosol MBP for expression test) construction
Double digest result shows that clone have failed. No band of right molecular weight can be detected.
MBP (cytosol MBP for mercury binding test) construction
Clones have been constructed, but need to be verified by double digestion.
Mercury Semiquantative Detection
Solution Preparation
1 g/L Dithizone Cloroform solution
1.038×10-3 mol/L HgCl2
Dithizone Eluant
Na2S detoxic solution
1 mol/L NaOH
Concentrated HNO3
Standard Mercury Concentration Determination
Mercury concentrated 25 times
Color change can be easily seen, but out of linear range of the standard curve.
7.12-7.18
DsbA-MBP (periplasmic MBP for expression test) construction
Band of expected molecular weight can be detected by double digest. Need to be sequenced.
Re-design the construction strategy for DsbA-MerR(MBP)
MBP-His & MBP construction
Failed.
Mercury Semiquantative Determination
Standard solution concentrated 10 times.
There is no significant difference between detected mercury concentration prepared in LB or water. But the absolute value is too low.
7.19-7.25
DsbA-MerR construction. Need to be verified.
DsbA-MBP construction. Need to be verified.
SDS-PAGE & Western Blot
Buffer preparation
5 × SDS-PAGE buffer
Tris – HCl pH 6.8
Tris – HCl pH 8.8
30% Acr / Bis-Acr
5 × TBS
5 × Transfer Buffer
1 × TTBS
SDS-PAGE result of DsbA-MerR
Overexpression band can be detected at the expected molecular weight (40 kD).
7.26-7.28 & 8.3-8.8
Double digest verification for DsbA-MBP, MBP-His & MBP.
Failed.
Check the template of MBP by sequencing.
Re-design for MBP & DsbA-MBP construction.
August
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 | 31 | - | - | - | - |
[TOP]
8.9-8.15
MBP-His construction
Sequencing.
MBP construction
Sequencing.
DsbA-MBP construction
strategy I
Sequencing.
Strategy II
Sequencing.
Standardization for DsbA-MerR
Sequencing.
Standardization for DsbA-MBP
DsbA-MerR sample preparation for SDS-PAGE
8.16-8.22
DsbA-MerR standardization finished.
Re-construct DsbA-MBP
Expression condition optimization for DsbA-MerR
8.23-8.29
SDS-PAGE & Western Blot for DsbA-MerR
SDS-PAGE & Western Blot for DsbA-MBP
The ratio of the primary antigen to secondary antigen should be optimized to get better figures.
8.30-9.5
SDS-PAGE for T3 polymerase
SDS-PAGE & Western Blot for MBP
The ratio of the primary antigen to secondary antigen should be optimized to get better figures.
September
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
- | - | 1 | 2 | 3 | 4 | 5 |
6 | 7 | 8 | 9 | 10 | 11 | 12 |
13 | 14 | 15 | 16 | 17 | 18 | 19 |
20 | 21 | 22 | 23 | 24 | 25 | 26 |
27 | 28 | 29 | 30 | - | - | - |
[TOP]
9.6-9.12
Western Blot for MBP
Western Blot for DsbA-MBP
9.13-9.19
SDS-PAGE & Western Blot for lead DsbA-MBP
SDS-PAGE for lead MBP
The molecular weight of the overexpression bands does not meet our expectation. Check the plasmid.
Scan the spectrum of Crt EBI
9.20-9.26
Characterize the mutation of PmerT.
9.27-10.3
1-18I-pag, 2-2M-pag, 1-18E-pag construction
October
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
- | - | - | - | 1 | 2 | 3 |
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | - | - | - | - |
[TOP]
10.4-10.10
western blot for mercury Lpp-OmpA-MBP
10.11-10.17
Cigarette digestion for our biokit.
Failed.